专利名称：重组悦目金蛛大壶状腺丝蛋白及其制备方法
技术领域：
本发明属于基因工程技术领域。
背景技术：
由于蜘蛛天然拖丝具有优于蚕丝的力学特性，除其强度能与蚕丝媲美外，最主要的优点是其同时具备蚕丝所缺乏的良好的弹性。优良的力学特性使蜘蛛拖丝在材料科学上有重要意义，尤其在某些特殊领域如医学、军事及航空航天等方面具有重要的应用价值。但由于蜘蛛独居和肉食的生活习性以及相互残杀的特点使人工大规模、高密度饲养蜘蛛以获得蛛丝的方案很难实施，而蜘蛛拖丝由大壶状腺丝蛋白分子组成，因此克隆大壶状腺丝蛋白基因进而通过基因工程方法生产重组大壶状腺丝蛋白成为首选途径。
发明内容
本发明需要解决的问题克隆悦目金蛛大壶状腺丝蛋白基因，并通过基因工程技术获得大壶状腺重组丝蛋白，为人工蜘蛛拖丝的生产提供原料。
本发明人所述悦目金蛛大壶状腺丝蛋白基因的序列是发明人在南京师范大学生命科学学院遗传资源研究所通过一系列的实验获得的。实验步骤和结果为1、根据其他蜘蛛大壶状腺丝蛋白的氨基酸序列设计简并引物PMaSp25’-GGNTANGGNCCNGGNTCNGG-3’(N＝A，T，CorT)，用悦目金蛛丝腺SMART RACE cDNA文库(图1)为模板，运用3′RACE技术获得了几条电泳条带(图2)，每个条带分别切胶纯化后序列测定。
2、根据1获得的cDNA序列测定的结果表明，其中一条大小约为900bp的条带被证明为悦目金蛛大壶状腺丝蛋白基因的3’端功能片段(图2)。测序后获得长度为918bp的大壶状腺丝蛋白基因(Aa-MaSp2)的cDNA序列(图3)，该序列由816bp的编码区(编码272个氨基酸)和99bp的3′端非翻译区组成，加尾信号AATAAA位于距离终止密码子(TAA)下游57bp处。美国国家生物技术情报中心(National Center for Biotechnology Information，NCBI)已正式接受该基因为GenBank的新成员，检索号为AY365017。
1 ggaggttatg gacccggatc tggacaacaa ggaccaggag gagccggtca acaaggacct61 ggaggtctag gaccatatgg acctggtgca gcagcagcag ccgcaggtag ctatggacca121 agagcaggcc aacagagacc aggagctggt caacaaggac ctggagctgg acaacaaggt181 ccaggtggag caggccaacg aggaccgtac ggacccgggg cagcagccgc agctgcagcc241 gctggcggct atggacaaca aggacctgga gctggacaac aaggaccagg agtaggacaa301 caaggtccag gtggagcagg ccaacgagga ccatacggac ccggggcagc agccgcagct361 gcagccgctg gtggctatgg accaagatat ggacaacaag gacctggagc aggaccatat421 gggccaggag caggagccac agctgctgcc gctggaggat atggaccagg agctggacag481 caaggaccaa gaagtcaggc tccagtcgca tccgctgcag ccgctcgact ttcttctcct541 caagccggtt ctagagtttc atctgctgtt tcaactttgg tatcgagtgg acctactaat601 cctgcatcac tttctaatgc tatcggtagc gttgtttcgc aggttagtgc aagcaatccc661 ggccttccta gttgcgatgt tcttgtgcaa gcattactgg aaatcgtatc cgccctcgta721 catatccttg gatcttccag cattggccaa attaactatt ccgcttcttc tcagtatgcc781 cgattagttg gccaatctat tgctcaggcc cttggttaat ttcataaatg atttcaattt841 ttctattaat gtaatgacag atttgtaatc tttctgaata aattttgaag catatttaaa901 aaaaaaaaaa aaaaaaaaa悦目金蛛大壶状腺丝蛋白基因cDNA序列Gly Gly Tyr Gly Pro Gly Ser Gly Gln Gln Gly Pro Gly Gly Ala Gly Gln Gln Gly Pro1 6 11 16Gly Gly Leu Gly Pro Tyr Gly Pro Gly Ala Ala Ala Ala Ala Ala Gly Ser Tyr Gly Pro21 26 31 36Arg Ala Gly Gln Gln Arg Pro Gly Ala Gly Gln Gln Gly Pro Gly Ala Gly Gln Gln Gly41 46 51 56Pro Gly Gly Ala Gly Gln Arg Gly Pro Tyr Gly Pro Gly Ala Ala Ala Ala Ala Ala Ala61 66 71 76Ala Gly Gly Tyr Gly Gln Gln Gly Pro Gly Ala Gly Gln Gln Gly Pro Gly Val Gly Gln81 86 91 96Gln Gly Pro Gly Gly Ala Gly Gln Arg Gly Pro Tyr Gly Pro Gly Ala Ala Ala Ala Ala101 106 111 116Ala Ala Ala Gly Gly Tyr Gly Pro Arg Tyr Gly Gln Gln Gly Pro Gly Ala Gly Pro Tyr121 126 131 136Gly Pro Gly Ala Gly Ala Thr Ala Ala Ala Ala Gly Gly Tyr Gly Pro Gly Ala Gly Gln141 146 151 156Gln Gly Pro Arg Ser Gln Ala Pro Val Ala Ser Ala Ala Ala Ala Arg Leu Ser Ser Pro161 165 171 176Gln Ala Gly Ser Arg Val Ser Ser Ala Val Ser Thr Leu Val Ser Ser Gly Pro Thr Asn181 186 191 196Pro Ala Ser Leu Ser Asn Ala Ile Gly Ser Val Val Ser Gln Val Ser Ala Ser Asn Pro201 206 211 216Gly Leu Pro Ser Cys Asp Val Leu Val Gln Ala Leu Leu Glu Ile Val Ser Ala Leu Val221 226 231 236
His Ile Leu Gly Ser Ser Ser Ile Gly Gln Ile Asn Tyr Ser Ala Ser Ser Gln Tyr Ala241 246 251 256Arg Leu Val Gly Gln Ser Ile Ala Gln Ala Leu Gly261 266 271悦目金蛛大壶状腺丝蛋白氨基酸序列3、由于蜘蛛大壶状腺丝蛋白基因大部分为重复序列，所以克隆该基因的长片段非常困难。为使通过基因工程手段获得的人工大壶状腺丝蛋白分子量尽量接近与蜘蛛自然产大壶状腺丝蛋白分子，我们利用2中得到的悦目金蛛大壶状腺丝蛋白基因cDNA序列中的功能片段设计了4个引物(primer-15’-GCGACGGGATCCGGAGGTTATGGACCC-3’；primer-25’-TAGCATGGATCCTACGACCATGGGAGGTTATGGACCC-3’；primer-35’-GCGATCAAGCTTGTACGAGATCTTGCTGCAGCGGCTGCAGCGGATGCGACTGGAGCC-3’；primer-45’-TACGTAAAGCTTTAATTTAGTGGTGGTGGTGGTGGTGACCAAGGGCCTGAGC-3’)，并采用基因操作技术(图4)人工拼接了编码悦目金蛛大壶状腺丝蛋白的人工基因-I和人工基因-II，这两个编码悦目金蛛大壶状腺丝蛋白的人工基因也被美国国家生物技术情报中心(National Center for BiotechnologyInformation，NCBI)所属GenBank确认，检索号分别为AY365013和AY365014。
1 ggatcctacg accatgggag gttatggacc cggatctgga caacaaggac caggaggagc61 cggtcaacaa ggacctggag gtctaggacc atatggacct ggtgcagcag cagcagccgc121 aggtagctat ggaccaagag caggccaaca gagaccagga gctggtcaac aaggacctgg181 agctggacaa caaggtccag gtggagcagg ccaacgagga ccgtacggac ccggggcagc241 agccgcagct gcagccgctg gcggctatgg acaacaagga cctggagctg gacaacaagg301 accaggagta ggacaacaag gtccaggtgg agcaggccaa cgaggaccat acggacccgg361 ggcagcagcc gcagctgcag ccgctggtgg ctatggacca agatatggac aacaaggacc421 tggagcagga ccatatgggc caggagcagg agccacagct gctgccgctg gaggatatgg481 accaggagct ggacagcaag gaccaagaag tcaggctcca gtcgcatccg ctgcagccgc541 tgcagcaaga tccggaggtt atggacccgg atctggacaa caaggaccag gaggagccgg601 tcaacaagga cctggaggtc taggaccata tggacctggt gcagcagcag cagccgcagg661 tagctatgga ccaagagcag gccaacagag accaggagct ggtcaacaag gacctggagc721 tggacaacaa ggtccaggtg gagcaggcca acgaggaccg tacggacccg gggcagcagc781 cgcagctgca gccgctggcg gctatggaca acaaggacct ggagctggac aacaaggacc841 aggagtagga caacaaggtc caggtggagc aggccaacga ggaccatacg gacccggggc901 agcagccgca gctgcagccg ctggtggcta tggaccaaga tatggacaac aaggacctgg961 agcaggacca tatgggccag gagcaggagc cacagctgct gccgctggag gatatggacc1021 aggagctgga cagcaaggac caagaagtca ggctccagtc gcatccgctg cagccgctcg1081 actttcttct cctcaagccg gttctagagt ttcatctgct gtttcaactt tggtatcgag1141 tggacctact aatcctgcat cactttctaa tgctatcggt agcgttgttt cgcaggttag1201 tgcaagcaat cccggccttc ctagttgcga tgttcttgtg caagcattac tggaaatcgt
1261 atccgccctc gtacatatcc ttggatcttc cagcattggc caaattaact attccgcttc1321 ttctcagtat gcccgattag ttggccaatc tattgctcag gcccttggtc accaccacca1381 ccaccactaa gtcgacatta aagctt编码悦目金蛛大壶状腺丝蛋白的人工基因-I DNA序列Gly Gly Tyr Gly Pro Gly Ser Gly Gln Gln Gly Pro Gly Gly Ala Gly Gln Gln Gly Pro1 6 11 16Gly Gly Leu Gly Pro Tyr Gly Pro Gly Ala Ala Ala Ala Ala Ala Gly Ser Tyr Gly Pro21 26 31 36Arg Ala Gly Gln Gln Arg Pro Gly Ala Gly Gln Gln Gly Pro Gly Ala Gly Gln Gln Gly41 46 51 56Pro Gly Gly Ala Gly Gln Arg Gly Pro Tyr Gly Pro Gly Ala Ala Ala Ala Ala Ala Ala61 66 71 76Ala Gly Gly Tyr Gly Gln Gln Gly Pro Gly Ala Gly Gln Gln Gly Pro Gly Val Gly Gln81 86 91 96Gln Gly Pro Gly Gly Ala Gly Gln Arg Gly Pro Tyr Gly Pro Gly Ala Ala Ala Ala Ala101 106 111 116Ala Ala Ala Gly Gly Tyr Gly Pro Arg Tyr Gly Gln Gln Gly Pro Gly Ala Gly Pro Tyr121 126 131 136Gly Pro Gly Ala Gly Ala Thr Ala Ala Ala Ala Gly Gly Tyr Gly Pro Gly Ala Gly Gln141 146 151 156Gln Gly Pro Arg Ser Gln Ala Pro Val Ala Ser Ala Ala Ala Ala Ala Ala Arg Ser Gly161 165 171 176Gly Tyr Gly Pro Gly Ser Gly Gln Gln Gly Pro Gly Gly Ala Gly Gln Gln Gly Pro Gly181 186 191 196Gly Leu Gly Pro Tyr Gly Pro Gly Ala Ala Ala Ala Ala Ala Gly Ser Tyr Gly Pro Arg201 206 211 216Ala Gly Gln Gln Arg Pro Gly Ala Gly Gln Gln Gly Pro Gly Ala Gly Gln Gln Gly Pro221 226 231 236Gly Gly Ala Gly Gln Arg Gly Pro Tyr Gly Pro Gly Ala Ala Ala Ala Ala Ala Ala Ala241 246 251 256Gly Gly Tyr Gly Gln Gln Gly Pro Gly Ala Gly Gln Gln Gly Pro Gly Val Gly Gln Gln261 266 271 276Gly Pro Gly Gly Ala Gly Gln Arg Gly Pro Tyr Gly Pro Gly Ala Ala Ala Ala Ala Ala281 286 291 296Ala Ala Gly Gly Tyr Gly Pro Arg Tyr Gly Gln Gln Gly Pro Gly Ala Gly Pro Tyr Gly301 306 311 316Pro Gly Ala Gly Ala Thr Ala Ala Ala Ala Gly Gly Tyr Gly Pro Gly Ala Gly Gln Gln321 326 331 336Gly Pro Arg Ser Gln Ala Pro Val Ala Ser Ala Ala Ala Ala Arg Leu Ser Ser Pro Gln341 346 351 356Ala Gly Ser Arg Val Ser Ser Ala Val Ser Thr Leu Val Ser Ser Gly Pro Thr Asn Pro
361 366 371 376Ala Ser Leu Ser Asn Ala Ile Gly Ser Val Val Ser Gln Val Ser Ala Ser Asn Pro Gly381 386 391 396Leu Pro Ser Cys Asp Val Leu Val Gln Ala Leu Leu Glu Ile Val Ser Ala Leu Val His401 406 411 416Ile Leu Gly Ser Ser Ser Ile Gly Gln Ile Asn Tyr Ser Ala Ser Ser Gln Tyr Ala Arg421 426 431 436Leu Val Gly Gln Ser Ile Ala Gln Ala Leu Gly His His His His His His441 446 451 456人工基因-I编码的悦目金蛛大壶状腺丝蛋白氨基酸序列1 ggatcctacg accatgggag gttatggacc cggatctgga caacaaggac caggaggagc61 cggtcaacaa ggacctggag gtctaggacc atatggacct ggtgcagcag cagcagccgc121 aggtagctat ggaccaagag caggccaaca gagaccagga gctggtcaac aaggacctgg181 agctggacaa caaggtccag gtggagcagg ccaacgagga ccgtacggac ccggggcagc241 agccgcagct gcagccgctg gcggctatgg acaacaagga cctggagctg gacaacaagg301 accaggagta ggacaacaag gtccaggtgg agcaggccaa cgaggaccat acggacccgg361 ggcagcagcc gcagctgcag ccgctggtgg ctatggacca agatatggac aacaaggacc421 tggagcagga ccatatgggc caggagcagg agccacagct gctgccgctg gaggatatgg481 accaggagct ggacagcaag gaccaagaag tcaggctcca gtcgcatccg ctgcagccgc541 tgcagcaaga tccggaggtt atggacccgg atctggacaa caaggaccag gaggagccgg601 tcaacaagga cctggaggtc taggaccata tggacctggt gcagcagcag cagccgcagg661 tagctatgga ccaagagcag gccaacagag accaggagct ggtcaacaag gacctggagc721 tggacaacaa ggtccaggtg gagcaggcca acgaggaccg tacggacccg gggcagcagc781 cgcagctgca gccgctggcg gctatggaca acaaggacct ggagctggac aacaaggacc841 aggagtagga caacaaggtc caggtggagc aggccaacga ggaccatacg gacccggggc901 agcagccgca gctgcagccg ctggtggcta tggaccaaga tatggacaac aaggacctgg961 agcaggacca tatgggccag gagcaggagc cacagctgct gccgctggag gatatggacc1021 aggagctgga cagcaaggac caagaagtca ggctccagtc gcatccgctg cagccgctgc1081 agcaagatcc ggaggttatg gacccggatc tggacaacaa ggaccaggag gagccggtca1141 acaaggacct ggaggtctag gaccatatgg acctggtgca gcagcagcag ccgcaggtag1201 ctatggacca agagcaggcc aacagagacc aggagctggt caacaaggac ctggagctgg1261 acaacaaggt ccaggtggag caggccaacg aggaccgtac ggacccgggg cagcagccgc1321 agctgcagcc gctggcggct atggacaaca aggacctgga gctggacaac aaggaccagg1381 agtaggacaa caaggtccag gtggagcagg ccaacgagga ccatacggac ccggggcagc1441 agccgcagct gcagccgctg gtggctatgg accaagatat ggacaacaag gacctggagc1501 aggaccatat gggccaggag caggagccac agctgctgcc gctggaggat atggaccagg1561 agctggacag caaggaccaa gaagtcaggc tccagtcgca tccgctgcag ccgctcgact1621 ttcttctcct caagccggtt ctagagtttc atctgctgtt tcaactttgg tatcgagtgg1681 acctactaat cctgcatcac tttctaatgc tatcggtagc gttgtttcgc aggttagtgc1741 aagcaatccc ggccttccta gttgcgatgt tcttgtgcaa gcattactgg aaatcgtatc1801 cgccctcgta catatccttg gatcttccag cattggccaa attaactatt ccgcttcttc
1861 tcagtatgcc cgattagttg gccaatctat tgctcaggcc cttggtcacc accaccacca1921 ccactaagtc gacattaaag ctt编码悦目金蛛大壶状腺丝蛋白的人工基因-II DNA序列Gly Gly Tyr Gly Pro Gly Ser Gly Gln Gln Gly Pro Gly Gly Ala Gly Gln Gln Gly Pro1 6 11 16Gly Gly Leu Gly Pro Tyr Gly Pro Gly Ala Ala Ala Ala Ala Ala Gly Ser Tyr Gly Pro21 26 31 36Arg Ala Gly Gln Gln Arg Pro Gly Ala Gly Gln Gln Gly Pro Gly Ala Gly Gln Gln Gly41 46 51 56Pro Gly Gly Ala Gly Gln Arg Gly Pro Tyr Gly Pro Gly Ala Ala Ala Ala Ala Ala Ala61 66 71 76Ala Gly Gly Tyr Gly Gln Gln Gly Pro Gly Ala Gly Gln Gln Gly Pro Gly Val Gly Gln81 86 91 96Gln Gly Pro Gly Gly Ala Gly Gln Arg Gly Pro Tyr Gly Pro Gly Ala Ala Ala Ala Ala101 106 111 116Ala Ala Ala Gly Gly Tyr Gly Pro Arg Tyr Gly Gln Gln Gly Pro Gly Ala Gly Pro Tyr121 126 131 136Gly Pro Gly Ala Gly Ala Thr Ala Ala Ala Ala Gly Gly Tyr Gly Pro Gly Ala Gly Gln141 146 151 156Gln Gly Pro Arg Ser Gln Ala Pro Val Ala Ser Ala Ala Ala Ala Ala Ala Arg Ser Gly161 165 171 176Gly Tyr Gly Pro Gly Ser Gly Gln Gln Gly Pro Gly Gly Ala Gly Gln Gln Gly Pro Gly181 186 191 196Gly Leu Gly Pro Tyr Gly Pro Gly Ala Ala Ala Ala Ala Ala Gly Ser Tyr Gly Pro Arg201 206 211 216Ala Gly Gln Gln Arg Pro Gly Ala Gly Gln Gln Gly Pro Gly Ala Gly Gln Gln Gly Pro221 226 231 236Gly Gly Ala Gly Gln Arg Gly Pro Tyr Gly Pro Gly Ala Ala Ala Ala Ala Ala Ala Ala241 246 251 256Gly Gly Tyr Gly Gln Gln Gly Pro Gly Ala Gly Gln Gln Gly Pro Gly Val Gly Gln Gln261 266 271 276Gly Pro Gly Gly Ala Gly Gln Arg Gly Pro Tyr Gly Pro Gly Ala Ala Ala Ala Ala Ala281 286 291 296Ala Ala Gly Gly Tyr Gly Pro Arg Tyr Gly Gln Gln Gly Pro Gly Ala Gly Pro Tyr Gly301 306 311 316Pro Gly Ala Gly Ala Thr Ala Ala Ala Ala Gly Gly Tyr Gly Pro Gly Ala Gly Gln Gln321 326 331 336Gly Pro Arg Ser Gln Ala Pro Val Ala Ser Ala Ala Ala Ala Ala Ala Arg Ser Gly Gly341 346 351 356Tyr Gly Pro Gly Ser Gly Gln Gln Gly Pro Gly Gly Ala Gly Gln Gln Gly Pro Gly Gly361 366 371 376
Leu Gly Pro Tyr Gly Pro Gly Ala Ala Ala Ala Ala Ala Gly Ser Tyr Gly Pro Arg Ala381 386 391 396Gly Gln Gln Arg Pro Gly Ala Gly Gln Gln Gly Pro Gly Ala Gly Gln Gln Gly Pro Gly401 406 411 416Gly Ala Gly Gln Arg Gly Pro Tyr Gly Pro Gly Ala Ala Ala Ala Ala Ala Ala Ala Gly421 426 431 436Gly Tyr Gly Gln Gln Gly Pro Gly Ala Gly Gln Gln Gly Pro Gly Val Gly Gln Gln Gly441 446 451 456Pro Gly Gly Ala Gly Gln Arg Gly Pro Tyr Gly Pro Gly Ala Ala Ala Ala Ala Ala Ala461 466 471 476Ala Gly Gly Tyr Gly Pro Arg Tyr Gly Gln Gln Gly Pro Gly Ala Gly Pro Tyr Gly Pro481 486 491 496Gly Ala Gly Ala Thr Ala Ala Ala Ala Gly Gly Tyr Gly Pro Gly Ala Gly Gln Gln Gly501 506 511 516Pro Arg Ser Gln Ala Pro Val Ala Ser Ala Ala Ala Ala Arg Leu Ser Ser Pro Gln Ala521 526 531 536Gly Ser Arg Val Ser Ser Ala Val Ser Thr Leu Val Ser Ser Gly Pro Thr Asn Pro Ala541 546 551 556Ser Leu Ser Asn Ala Ile Gly Ser Val Val Ser Gln Val Ser Ala Ser Asn Pro Gly Leu561 566 571 576Pro Ser Cys Asp Val Leu Val Gln Ala Leu Leu Glu Ile Val Ser Ala Leu Val His Ile581 585 591 596Leu Gly Ser Ser Ser Ile Gly Gln Ile Asn Tyr Ser Ala Ser Ser Gln Tyr Ala Arg Leu601 606 611 616Val Gly Gln Ser Ile Ala Gln Ala Leu Gly His His His His His His621 626 631 636人工基因-II编码的悦目金蛛大壶状腺丝蛋白氨基酸序列本发明技术方案l、悦目金蛛大壶状腺丝蛋白的制备(1)以提取的悦目金蛛丝腺总RNA为模板，用SMARTTMcDNA Library Construction Kit试剂盒(购自Clontech公司)反转录合成悦目金蛛丝腺SMART RACE cDNA文库。利用简便引物PMaSp25’-GGNTANGGNCCNGGNTCNGG-3’(N＝A，T，CorT)和试剂盒提供的接头引物3’PCRPrimer(5’-TTCTAGAGGCCGAGGCGGCC-3’)进行3’RACE，把PCR产物切胶纯化后连接于pMDl8-T载体(购自Takara公司)，通过序列测定鉴定后获得含有悦目金蛛大壶状腺丝蛋白基因的重组质粒pMD18-T-MaSp2。
(2)用引物5’-GCACGACCATGGGAGGTTATGGACCC-3’和5’-TAATGGATCCTTAGTGGTGGTGGTGGTGGTGACCAAGGGCCTGAGCA-3’以上述重组质粒pMDl8-T-MaSp2为模板扩增出悦目金蛛大壶状腺丝蛋白基因的编码区。反应条件95℃预变性5min，95℃变性30s，60℃退火30s，72℃延伸1min，30个循环，72℃延伸10min。
(3)上述扩增产物割胶纯化后与表达载体pET28 vector(购自Novagen公司)同时用NcoI和BamHI在37℃双酶切数小时。琼脂糖凝胶电泳回收酶切片段及pET28载体后用T4DNA连接酶连接。将连接产物转化大肠杆菌DH5α，通过双酶切鉴定重组表达质粒pET28-MaSp2(图5)。
(4)将重组质粒pET28-MaSp2转化大肠杆菌BL21(DE3)菌株后，以IPTG诱导若干小时，离心后加上样缓冲液煮沸破碎细胞，进行12％浓度的SDS-PAGE电泳(图6)。
(5)利用Ni-NTA His-bind亲和层析柱(购自Novagen公司)对重组蛋白进行初步分离纯化，然后用分子筛技术进行二次纯化。
2、悦目金蛛大壶状腺丝蛋白多克隆抗体的制备将重组蛋白与佐剂(Complete/Incomplete Freund’s Adjuvant)等量混合，免疫新西兰兔(背部皮下注射)，每隔3-4周激发注射一次，共三次，于3个月后取血分离血清。第一次免疫使用完全佐剂，后几次免疫使用不完全佐剂。以免疫前动物血清作为对照做ELISA，测定各免疫兔血清中抗体滴度。筛选高滴度血清，进一步用抗原作竞争抑制免疫试验确定抗体的特异性。
3、编码大壶状腺丝蛋白的人工基因-I和人工基因-II的表达(1)在通过基因操作技术拼接编码大壶状腺丝蛋白的人工基因-I和人工基因-II的过程中(图4)，这两个人工基因已被设计与表达质粒pET28结合，形成的重组表达质粒分别为pET28-MaSp2(2+1)和pET28-MaSp2(3+1)。
(2)把重组表达质粒pET28-MaSp2(2+1)和pET28-MaSp2(3+1)分别转化大肠杆菌BL21(DE3)菌株后，以IPTG诱导若干小时，离心后加上样缓冲液煮沸破碎细胞，进行12％浓度的SDS-PAGE电泳(图7，8)。
(3)利用Ni-NTA His-bind亲和层析柱(购自Novagen公司)对重组蛋白进行初步分离纯化，然后用分子筛技术进行二次纯化。
4、重组大壶状腺丝蛋白的鉴定(1)把纯化后的三种重组丝蛋白MaSp2，MaSp2(2+1)和MaSp2(3+1)分别于110℃下用浓盐酸水解24小时，再在日立835-50型氨基酸分析仪上分析它们的氨基酸组成和含量。
(2)把纯化后的三种重组丝蛋白MaSp2，MaSp2(2+1)和MaSp2(3+1)分别进行12％浓度的SDS-PAGE电泳，再通过蛋白质转移的方法转至PVDF膜上，在ProciseTM491型蛋白质序列测定仪(购自PE/ABI公司)上分别测定每种重组丝蛋白的N端10个氨基酸残基的序列。
(3)纯化后的三种重组丝蛋白MaSp2，MaSp2(2+1)和MaSp2(3+1)分别进行12％浓度的SDS-PAGE电泳，再通过蛋白质转移的方法转至PVDF膜上，利用悦目金蛛大壶状腺丝蛋白多克隆抗体进行Western分析以鉴定重组丝蛋白(图9)。
5、保持重组大壶状腺丝蛋白稳定溶解性的缓冲液体系的筛选及人工拖丝的研制由于大壶状腺丝蛋白分子有许多连续的Ala残基组成的重复片断，从而具有自发形成不溶于水的β-折叠二级结构的倾向。但根据蜘蛛自然蛛丝形成机理，丝蛋白在从纺孔排出体外、转变为固态丝之前始终处于浓蛋白水溶液的状态，所以筛选合适的、能高浓度溶解重组丝蛋白的缓冲液体系非常必要。用饱和硫酸氨沉淀重组丝蛋白后用6M盐酸胍溶解丝蛋白沉淀，再用不同参数的缓冲液进行透析以观察丝蛋白在各种缓冲液中的溶解性。通过这种方法已经筛选到PH值为2-4的一种缓冲液体系(50mmol/L NaCl；3.0mmol/L KCll0mmol/L Na2HPO4；3mmol/KH2PO4)能溶解20-30％的重组丝蛋白，从而解决了人工拖丝研制中保持重组丝蛋白在水中溶解性这个关键问题。人工拖丝可开发成为一种在材料科学、医学、军事及航空航天等领域有应用价值的可降解的新型生物材料。
四
图1、悦目金蛛丝腺SMART RACE cDNA文库电泳图(MDNA标记；1双链cDNA)图2、3’RACE PCR结果(MDNA标记；l3’RACE PCR产物；箭头所指条带为目的条带)图3、悦目金蛛大壶状腺丝蛋白基因cDNA序列及编码蛋白的氨基酸序列图4、编码大壶状腺丝蛋白的人工基因-I和人工基因-II的拼接过程示意5、重组表达质粒的限制性酶切分析(MDNA分子量标记；1pET28-MaSp2；2pET28-MaSp2经NcoI和BamHI双酶切；3pET28-MaSp2(2+1)；4pET28-MaSp2(2+1)经NcoI和HindIII双酶切；5pET28-MaSp2(3+1)；6pET28-MaSp2(3+1)经NcoI和HindIII双酶切)图6、悦目金蛛大壶状腺丝蛋白诱导表达及蛋白质纯化的聚丙烯酰氨凝胶电泳分析(1BL21(DE3)+pET28；2BL21(DE3)+pET28-MaSp2经IPTG诱导0小时；3BL21(DE3)+pET28-MaSp2经IPTG诱导4小时；4蛋白质分子量标记；5BL21(DE3)+pET28-MaSp2经IPTG诱导4小时后细菌破碎后的上清液；6BL21(DE3)+pET28-MaSp2经IPTG诱导4小时后细菌破碎后的沉淀；7重组蛋白经Ni-NTA His-bind亲和层析柱初步纯化；8重组蛋白经分子筛技术进行二次纯化)图7、人工基因-I诱导表达及蛋白质纯化的聚丙烯酰氨凝胶电泳分析(1BL21(DE3)+pET28；2BL21(DE3)+pET28-MaSp2(2+1)经IPTG诱导0小时；3BL21(DE3)+pET28-MaSp2(2+1)经IPTG诱导4小时；4蛋白质分子量标记；5BL21(DE3)+pET28-MaSp2(2+1)经IPTG诱导4小时后细菌破碎后的上清液；6BL21(DE3)+pET28-MaSp2(2+1)经IPTG诱导4小时后细菌破碎后的沉淀；7重组蛋白经Ni-NTA His-bind亲和层析柱初步纯化；8重组蛋白经分子筛技术进行二次纯化)图8、人工基因-II诱导表达及蛋白质纯化的聚丙烯酰氨凝胶电泳分析(1BL21(DE3)+pET28；2BL21(DE3)+pET28-MaSp2(3+1)经IPTG诱导0小时；3BL21(DE3)+pET28-MaSp2(3+1)经IPTG诱导4小时；4蛋白质分子量标记；5BL21(DE3)+pET28-MaSp2(3+1)经IPTG诱导4小时后细菌破碎后的上清液；6BL21(DE3)+pET28-MaSp2(3+1)经IPTG诱导4小时后细菌破碎后的沉淀；7重组蛋白经Ni-NTA His-bind亲和层析柱初步纯化；8重组蛋白经分子筛技术进行二次纯化)图9、重组大壶状腺丝蛋白的Western分析(1蛋白质分子量标记；2MaSp2；3MaSp2(2+1)；4MaSp2(3+1)；5阴性对照)
悦目金蛛大壶状腺丝蛋白基因序列表<110>南京师范大学<120>悦目金蛛大壶状腺丝蛋白基因(Aa-MaSp2)<160>3<210>1<211>918bp<212>cDNA<213>悦目金蛛(Argiope amoena)<400>10 ggaggttatg gacccggatc tggacaacaa ggaccaggag gagccggtca acaaggacct61 ggaggtctag gaccatatgg acctggtgca gcagcagcag ccgcaggtag ctatggacca121 agagcaggcc aacagagacc aggagctggt caacaaggac ctggagctgg acaacaaggt181 ccaggtggag caggccaacg aggaccgtac ggacccgggg cagcagccgc agctgcagcc241 gctggcggct atggacaaca aggacctgga gctggacaac aaggaccagg agtaggacaa301 caaggtccag gtggagcagg ccaacgagga ccatacggac ccggggcagc agccgcagct361 gcagccgctg gtggctatgg accaagatat ggacaacaag gacctggagc aggaccatat421 gggccaggag caggagccac agctgctgcc gctggaggat atggaccagg agctggacag481 caaggaccaa gaagtcaggc tccagtcgca tccgctgcag ccgctcgact ttcttctcct541 caagccggtt ctagagtttc atctgctgtt tcaactttgg tatcgagtgg acctactaat601 cctgcatcac tttctaatgc tatcggtagc gttgtttcgc aggttagtgc aagcaatccc661 ggccttccta gttgcgatgt tcttgtgcaa gcattactgg aaatcgtatc cgccctcgta721 catatccttg gatcttccag cattggccaa attaactatt ccgcttcttc tcagtatgcc781 cgattagttg gccaatctat tgctcaggcc cttggttaat ttcataaatg atttcaattt841 ttctattaat gtaatgacag atttgtaatc tttctgaata aattttgaag catatttaaa901 aaaaaaaaaa aaaaaaaaa悦目金蛛大壶状腺丝蛋白基因cDNA序列Gly Gly Tyr Gly Pro Gly Ser Gly Gln Gln Gly Pro Gly Gly Ala Gly Gln Gln Gly Pro1 6 11 16Gly Gly Leu Gly Pro Tyr Gly Pro Gly Ala Ala Ala Ala Ala Ala Gly Ser Tyr Gly Pro21 26 31 36
Arg Ala Gly Gln Gln Arg Pro Gly Ala Gly Gln Gln Gly Pro Gly Ala Gly Gln Gln Gly41 46 51 56Pro Gly Gly Ala Gly Gln Arg Gly Pro Tyr Gly Pro Gly Ala Ala Ala Ala Ala Ala Ala61 66 71 76Ala Gly Gly Tyr Gly Gln Gln Gly Pro Gly Ala Gly Gln Gln Gly Pro Gly Val Gly Gln81 86 91 96Gln Gly Pro Gly Gly Ala Gly Gln Arg Gly Pro Tyr Gly Pro Gly Ala Ala Ala Ala Ala101 106 111 116Ala Ala Ala Gly Gly Tyr Gly Pro Arg Tyr Gly Gln Gln Gly Pro Gly Ala Gly Pro Tyr121 126 131 136Gly Pro Gly Ala Gly Ala Thr Ala Ala Ala Ala Gly Gly Tyr Gly Pro Gly Ala Gly Gln141 146 151 156Gln Gly Pro Arg Ser Gln Ala Pro Val Ala Ser Ala Ala Ala Ala Arg Leu Ser Ser Pro161 165 171 176Gln Ala Gly Ser Arg Val Ser Ser Ala Val Ser Thr Leu Val Ser Ser Gly Pro Thr Asn181 186 191 196Pro Ala Ser Leu Ser Asn Ala Ile Gly Ser Val Val Ser Gln Val Ser Ala Ser Asn Pro201 206 211 216Gly Leu Pro Ser Cys Asp Val Leu Val Gln Ala Leu Leu Glu Ile Val Ser Ala Leu Val221 226 231 236His Ile Leu Gly Ser Ser Ser Ile Gly Gln Ile Asn Tyr Ser Ala Ser Ser Gln Tyr Ala241 246 251 256Arg Leu Val Gly Gln Ser Ile Ala Gln Ala Leu Gly261 266 271悦目金蛛大壶状腺丝蛋白氨基酸序列编码悦目金蛛大壶状腺丝蛋白的人工基因I序列表<110>南京师范大学<120>编码悦目金蛛大壶状腺丝蛋白的人工基因I<160>3<210>2
<21l>1406bp<212>DNA<213>人工序列<400>10 ggatcctacg accatgggag gttatggacc cggatctgga caacaaggac caggaggagc61 cggtcaacaa ggacctggag gtctaggacc atatggacct ggtgcagcag cagcagccgc121 aggtagctat ggaccaagag caggccaaca gagaccagga gctggtcaac aaggacctgg181 agctggacaa caaggtccag gtggagcagg ccaacgagga ccgtacggac ccggggcagc241 agccgcagct gcagccgctg gcggctatgg acaacaagga cctggagctg gacaacaagg301 accaggagta ggacaacaag gtccaggtgg agcaggccaa cgaggaccat acggacccgg361 ggcagcagcc gcagctgcag ccgctggtgg ctatggacca agatatggac aacaaggacc421 tggagcagga ccatatgggc caggagcagg agccacagct gctgccgctg gaggatatgg481 accaggagct ggacagcaag gaccaagaag tcaggctcca gtcgcatccg ctgcagccgc541 tgcagcaaga tccggaggtt atggacccgg atctggacaa caaggaccag gaggagccgg601 tcaacaagga cctggaggtc taggaccata tggacctggt gcagcagcag cagccgcagg661 tagctatgga ccaagagcag gccaacagag accaggagct ggtcaacaag gacctggagc721 tggacaacaa ggtccaggtg gagcaggcca acgaggaccg tacggacccg gggcagcagc781 cgcagctgca gccgctggcg gctatggaca acaaggacct ggagctggac aacaaggacc841 aggagtagga caacaaggtc caggtggagc aggccaacga ggaccatacg gacccggggc901 agcagccgca gctgcagccg ctggtggcta tggaccaaga tatggacaac aaggacctgg961 agcaggacca tatgggccag gagcaggagc cacagctgct gccgctggag gatatggacc1021 aggagctgga cagcaaggac caagaagtca ggctccagtc gcatccgctg cagccgctcg1081 actttcttct cctcaagccg gttctagagt ttcatctgct gtttcaactt tggtatcgag1141 tggacctact aatcctgcat cactttctaa tgctatcggt agcgttgttt cgcaggttag1201 tgcaagcaat cccggccttc ctagttgcga tgttcttgtg caagcattac tggaaatcgt1261 atccgccctc gtacatatcc ttggatcttc cagcattggc caaattaact attccgcttc1321 ttctcagtat gcccgattag ttggccaatc tattgctcag gcccttggtc accaccacca1381 ccaccactaa gtcgacatta aagctt编码悦目金蛛大壶状腺丝蛋白的人工基因-I DNA序列Gly Gly Tyr Gly Pro Gly Ser Gly Gln Gln Gly Pro Gly Gly Ala Gly Gln Gln Gly Prol 6 11 16
Gly Gly Leu Gly Pro Tyr Gly Pro Gly Ala Ala Ala Ala Ala Ala Gly Ser Tyr Gly Pro21 26 31 36Arg Ala Gly Gln Gln Arg Pro Gly Ala Gly Gln Gln Gly Pro Gly Ala Gly Gln Gln Gly41 46 51 56Pro Gly Gly Ala Gly Gln Arg Gly Pro Tyr Gly Pro Gly Ala Ala Ala Ala Ala Ala Ala61 66 71 76Ala Gly Gly Tyr Gly Gln Gln Gly Pro Gly Ala Gly Gln Gln Gly Pro Gly Val Gly Gln81 86 91 96Gln Gly Pro Gly Gly Ala Gly Gln Arg Gly Pro Tyr Gly Pro Gly Ala Ala Ala Ala Ala101 106 111 116Ala Ala Ala Gly Gly Tyr Gly Pro Arg Tyr Gly Gln Gln Gly Pro Gly Ala Gly Pro Tyr121 126 131 136Gly Pro Gly Ala Gly Ala Thr Ala Ala Ala Ala Gly Gly Tyr Gly Pro Gly Ala Gly Gln141 146 151 156Gln Gly Pro Arg Ser Gln Ala Pro Val Ala Ser Ala Ala Ala Ala Ala Ala Arg Ser Gly161 165 171 176Gly Tyr Gly Pro Gly Ser Gly Gln Gln Gly Pro Gly Gly Ala Gly Gln Gln Gly Pro Gly181 186 191 196Gly Leu Gly Pro Tyr Gly Pro Gly Ala Ala Ala Ala Ala Ala Gly Ser Tyr Gly Pro Arg201 206 211216Ala Gly Gln Gln Arg Pro Gly Ala Gly Gln Gln Gly Pro Gly Ala Gly Gln Gln Gly Pro221 226 231 236Gly Gly Ala Gly Gln Arg Gly Pro Tyr Gly Pro Gly Ala Ala Ala Ala Ala Ala Ala Ala241 246 251 256Gly Gly Tyr Gly Gln Gln Gly Pro Gly Ala Gly Gln Gln Gly Pro Gly Val Gly Gln Gln261 266 271 276Gly Pro Gly Gly Ala Gly Gln Arg Gly Pro Tyr Gly Pro Gly Ala Ala Ala Ala Ala Ala281 286 291 296Ala Ala Gly Gly Tyr Gly Pro Arg Tyr Gly Gln Gln Gly Pro Gly Ala Gly Pro Tyr Gly301 306 311 316Pro Gly Ala Gly Ala Thr Ala Ala Ala Ala Gly Gly Tyr Gly Pro Gly Ala Gly Gln Gln321 326 331 336Gly Pro Arg Ser Gln Ala Pro Val Ala Ser Ala Ala Ala Ala Arg Leu Ser Ser Pro Gln341 346 351 356
Ala Gly Ser Arg Val Ser Ser Ala Val Ser Thr Leu Val Ser Ser Gly Pro Thr Asn Pro361 366 371 376Ala Ser Leu Ser Asn Ala Ile Gly Ser Val Val Ser Gln Val Ser Ala Ser Asn Pro Gly381 386 391 396Leu Pro Ser Cys Asp Val Leu Val Gln Ala Leu Leu Glu Ile Val Ser Ala Leu Val His401 406 411 416Ile Leu Gly Ser Ser Ser Ile Gly Gln Ile Asn Tyr Ser Ala Ser Ser Gln Tyr Ala Arg421 426 431 436Leu Val Gly Gln Ser Ile Ala Gln Ala Leu Gly His His His His His His441 446 451 456人工基因-I编码的悦目金蛛大壶状腺丝蛋白氨基酸序列编码悦目金蛛大壶状腺丝蛋白的人工基因II序列表<110>南京师范大学<120>编码悦目金蛛大壶状腺丝蛋白的人工基因II<160>3<210>3<211>1943bp<212>DNA<213>人工序列<400>11 ggatcctacg accatgggag gttatggacc cggatctgga caacaaggac caggaggagc61 cggtcaacaa ggacctggag gtctaggacc atatggacct ggtgcagcag cagcagccgc121 aggtagctat ggaccaagag caggccaaca gagaccagga gctggtcaac aaggacctgg181 agctggacaa caaggtccag gtggagcagg ccaacgagga ccgtacggac ccggggcagc241 agccgcagct gcagccgctg gcggctatgg acaacaagga cctggagctggacaacaagg301 accaggagta ggacaacaag gtccaggtgg agcaggccaa cgaggaccat acggacccgg361 ggcagcagcc gcagctgcag ccgctggtgg ctatggacca agatatggac aacaaggacc421 tggagcagga ccatatgggc caggagcagg agccacagct gctgccgctg gaggatatgg481 accaggagct ggacagcaag gaccaagaag tcaggctcca gtcgcatccg ctgcagccgc541 tgcagcaaga tccggaggtt atggacccgg atctggacaa caaggaccag gaggagccgg
601 tcaacaagga cctggaggtc taggaccata tggacctggt gcagcagcag cagccgcagg661 tagctatgga ccaagagcag gccaacagag accaggagct ggtcaacaag gacctggagc721 tggacaacaa ggtccaggtg gagcaggcca acgaggaccg tacggacccg gggcagcagc781 cgcagctgca gccgctggcg gctatggaca acaaggacct ggagctggac aacaaggacc841 aggagtagga caacaaggtc caggtggagc aggccaacga ggaccatacg gacccggggc901 agcagccgca gctgcagccg ctggtggcta tggaccaaga tatggacaac aaggacctgg961 agcaggacca tatgggccag gagcaggagc cacagctgct gccgctggag gatatggacc1021 aggagctgga cagcaaggac caagaagtca ggctccagtc gcatccgctg cagccgctgc1081 agcaagatcc ggaggttatg gacccggatc tggacaacaa ggaccaggag gagccggtca1141 acaaggacct ggaggtctag gaccatatgg acctggtgca gcagcagcag ccgcaggtag1201 ctatggacca agagcaggcc aacagagacc aggagctggt caacaaggac ctggagctgg1261 acaacaaggt ccaggtggag caggccaacg aggaccgtac ggacccgggg cagcagccgc1321 agctgcagcc gctggcggct atggacaaca aggacctgga gctggacaac aaggaccagg1381 agtaggacaa caaggtccag gtggagcagg ccaacgagga ccatacggac ccggggcagc1441 agccgcagct gcagccgctg gtggctatgg accaagatat ggacaacaag gacctggagc1501 aggaccatat gggccaggag caggagccac agctgctgcc gctggaggat atggaccagg1561 agctggacag caaggaccaa gaagtcaggc tccagtcgca tccgctgcag ccgctcgact1621 ttcttctcct caagccggtt ctagagtttc atctgctgtt tcaactttgg tatcgagtgg1681 acctactaat cctgcatcac tttctaatgc tatcggtagc gttgtttcgc aggttagtgc1741 aagcaatccc ggccttccta gttgcgatgt tcttgtgcaagcattactggaaatcgtatc1801 cgccctcgta catatccttg gatcttccag cattggccaa attaactatt ccgcttcttc1861 tcagtatgcc cgattagttg gccaatctat tgctcaggcc cttggtcacc accaccacca1921 ccactaagtc gacattaaag ctt编码悦目金蛛大壶状腺丝蛋白的人工基因-II DNA序列Gly Gly Tyr Gly Pro Gly Ser Gly Gln Gln Gly Pro Gly Gly Ala Gly Gln Gln Gly Pro1 6 11 16Gly Gly Leu Gly Pro Tyr Gly Pro Gly Ala Ala Ala Ala Ala Ala Gly Ser Tyr Gly Pro21 26 31 36Arg Ala Gly Gln Gln Arg Pro Gly Ala Gly Gln Gln Gly Pro Gly Ala Gly Gln Gln Gly41 46 51 56Pro Gly Gly Ala Gly Gln Arg Gly Pro Tyr Gly Pro Gly Ala Ala Ala Ala Ala Ala Ala
61 66 71 76Ala Gly Gly Tyr Gly Gln Gln Gly Pro Gly Ala Gly Gln Gln Gly Pro Gly Val Gly Gln81 86 91 96Gln Gly Pro Gly Gly Ala Gly Gln Arg Gly Pro Tyr Gly Pro Gly Ala Ala Ala Ala Ala101 106 111 116Ala Ala Ala Gly Gly Tyr Gly Pro Arg Tyr Gly Gln Gln Gly Pro Gly Ala Gly Pro Tyr121 126 131 136Gly Pro Gly Ala Gly Ala Thr Ala Ala Ala Ala Gly Gly Tyr Gly Pro Gly Ala Gly Gln141 146 151 156Gln Gly Pro Arg Ser Gln Ala Pro Val Ala Ser Ala Ala Ala Ala Ala Ala Arg Ser Gly161 165 171 176Gly Tyr Gly Pro Gly Ser Gly Gln Gln Gly Pro Gly Gly Ala Gly Gln Gln Gly Pro Gly181 186 191 196Gly Leu Gly Pro Tyr Gly Pro Gly Ala Ala Ala Ala Ala Ala Gly Ser Tyr Gly Pro Arg201 206 211 216Ala Gly Gln Gln Arg Pro Gly Ala Gly Gln Gln Gly Pro Gly Ala Gly Gln Gln Gly Pro221 226 231 236Gly Gly Ala Gly Gln Arg Gly Pro Tyr Gly Pro Gly Ala Ala Ala Ala Ala Ala Ala Ala241 246 251 256Gly Gly Tyr Gly Gln Gln Gly Pro Gly Ala Gly Gln Gln Gly Pro Gly Val Gly Gln Gln261 266 271 276Gly Pro Gly Gly Ala Gly Gln Arg Gly Pro Tyr Gly Pro Gly Ala Ala Ala Ala Ala Ala281 286 291 296Ala Ala Gly Gly Tyr Gly Pro Arg Tyr Gly Gln Gln Gly Pro Gly Ala Gly Pro Tyr Gly301 306 311 316Pro Gly Ala Gly Ala Thr Ala Ala Ala Ala Gly Gly Tyr Gly Pro Gly Ala Gly Gln Gln321 326 331 336Gly Pro Arg Ser Gln Ala Pro Val Ala Ser Ala Ala Ala Ala Ala Ala Arg Ser Gly Gly341 346 351 356Tyr Gly Pro Gly Ser Gly Gln Gln Gly Pro Gly Gly Ala Gly Gln Gln Gly Pro Gly Gly361 366 371 376Leu Gly Pro Tyr Gly Pro Gly Ala Ala Ala Ala Ala Ala Gly Ser Tyr Gly Pro Arg Ala381 386 391 396Gly Gln Gln Arg Pro Gly Ala Gly Gln Gln Gly Pro Gly Ala Gly Gln Gln Gly Pro Gly
401 406 411 416Gly Ala Gly Gln Arg Gly Pro Tyr Gly Pro Gly Ala Ala Ala Ala Ala Ala Ala Ala Gly421 426 431 436Gly Tyr Gly Gln Gln Gly Pro Gly Ala Gly Gln Gln Gly Pro Gly Val Gly Gln Gln Gly441 446 451 456Pro Gly Gly Ala Gly Gln Arg Gly Pro Tyr Gly Pro Gly Ala Ala Ala Ala Ala Ala Ala461 466 471 476Ala Gly Gly Tyr Gly Pro Arg Tyr Gly Gln Gln Gly Pro Gly Ala Gly Pro Tyr Gly Pro481 486 491 496Gly Ala Gly Ala Thr Ala Ala Ala Ala Gly Gly Tyr Gly Pro Gly Ala Gly Gln Gln Gly501 506 511 516Pro Arg Ser Gln Ala Pro Val Ala Ser Ala Ala Ala Ala Arg Leu Ser Ser Pro Gln Ala521 526 531 536Gly Ser Arg Val Ser Ser Ala Val Ser Thr Leu Val Ser Ser Gly Pro Thr Asn Pro Ala541 546 551 556Ser Leu Ser Asn Ala Ile Gly Ser Val Val Ser Gln Val Ser Ala Ser Asn Pro Gly Leu561 566 571 576Pro Ser Cys Asp Val Leu Val Gln Ala Leu Leu Glu Ile Val Ser Ala Leu Val His Ile581 585 591 596Leu Gly Ser Ser Ser Ile Gly Gln Ile Asn Tyr Ser Ala Ser Ser Gln Tyr Ala Arg Leu601 606 611 616Val Gly Gln Ser Ile Ala Gln Ala Leu Gly His His His His His His621 626 631 636人工基因-II编码的悦目金蛛大壶状腺丝蛋白氨基酸序列
权利要求
1.一种悦目金蛛大壶状腺丝蛋白基因克隆制备的重组蛋白，其氨基酸序列为Gly Gly Tyr Gly Pro Gly Ser Gly Gln Gln Gly Pro Gly Gly Ala Gly Gln Gln Gly Pro1 6 11 16Gly Gly Leu Gly Pro Tyr Gly Pro Gly Ala Ala Ala Ala Ala Ala Gly Ser Tyr Gly Pro21 26 31 36Arg Ala Gly Gln Gln Arg Pro Gly Ala Gly Gln Gln Gly Pro Gly Ala Gly Gln Gln Gly41 46 51 56Pro Gly Gly Ala Gly Gln Arg Gly Pro Tyr Gly Pro Gly Ala Ala Ala Ala Ala Ala Ala61 66 71 76Ala Gly Gly Tyr Gly Gln Gln Gly Pro Gly Ala Gly Gln Gln Gly Pro Gly Val Gly Gln81 86 91 96Gln Gly Pro Gly Gly Ala Gly Gln Arg Gly Pro Tyr Gly Pro Gly Ala Ala Ala Ala Ala101 106 111 116Ala Ala Ala Gly Gly Tyr Gly Pro Arg Tyr Gly Gln Gln Gly Pro Gly Ala Gly Pro Tyr121 126 131 136Gly Pro Gly Ala Gly Ala Thr Ala Ala Ala Ala Gly Gly Tyr Gly Pro Gly Ala Gly Gln141 146 151 156Gln Gly Pro Arg Ser Gln Ala Pro Val Ala Ser Ala Ala Ala Ala Arg Leu Ser Ser Pro161 165 171 176Gln Ala Gly Ser Arg Val Ser Ser Ala Val Ser Thr Leu Val Ser Ser Gly Pro Thr Asn181 186 191 196Pro Ala Ser Leu Ser Asn Ala Ile Gly Ser Val Val Ser Gln Val Ser Ala Ser Asn Pro201 206 211 216Gly Leu Pro Ser Cys Asp Val Leu Val Gln Ala Leu Leu Glu Ile Val Ser Ala Leu Val221 226 231 236His Ile Leu Gly Ser Ser Ser Ile Gly Gln Ile Asn Tyr Ser Ala Ser Ser Gln Tyr Ala241 246 251 256Arg Leu Val Gly Gln Ser Ile Ala Gln Ala Leu Gly261 266 271
2.根据权利要求书1所述悦目金蛛大壶状腺丝蛋白的制备方法，其特征是以悦目金蛛大壶状腺丝蛋白基因(Aa-MaSp2)编码区段的核苷酸序列与质粒pET28制备成pET28-MaSp2表达质粒，在无NaCL的LB中30-35℃培养到OD600为0.5，经IPTG诱导，破碎细胞后经Ni-NTAHis-bind亲和层析柱和分子筛两次纯化后，得到纯化的重组蛋白。
3.根据权利要求书1所述，针对Aa-MaSp2基因开放阅读框氨基酸序列制备的多克隆抗体，其特征是融合蛋白与完全和不完全佐剂混合后，免疫新西兰兔制备多克隆抗体。
4.一种编码悦目金蛛大壶状腺丝蛋白的人工基因-I重组蛋白氨基酸序列为Gly Gly Tyr Gly Pro Gly Ser Gly Gln Gln Gly Pro Gly Gly Ala Gly Gln Gln Gly Pro1 6 11 16Gly Gly Leu Gly Pro Tyr Gly Pro Gly Ala Ala Ala Ala Ala Ala Gly Ser Tyr Gly Pro21 26 31 36Arg Ala Gly Gln Gln Arg Pro Gly Ala Gly Gln Gln Gly Pro Gly Ala Gly Gln Gln Gly41 46 51 56Pro Gly Gly Ala Gly Gln Arg Gly Pro Tyr Gly Pro Gly Ala Ala Ala Ala Ala Ala Ala61 66 71 76Ala Gly Gly Tyr Gly Gln Gln Gly Pro Gly Ala Gly Gln Gln Gly Pro Gly Val Gly Gln81 86 91 96Gln Gly Pro Gly Gly Ala Gly Gln Arg Gly Pro Tyr Gly Pro Gly Ala Ala Ala Ala Ala101 106 111 116Ala Ala Ala Gly Gly Tyr Gly Pro Arg Tyr Gly Gln Gln Gly Pro Gly Ala Gly Pro Tyr121 126 131 136Gly Pro Gly Ala Gly Ala Thr Ala Ala Ala Ala Gly Gly Tyr Gly Pro Gly Ala Gly Gln141 146 151 156Gln Gly Pro Arg Ser Gln Ala Pro Val Ala Ser Ala Ala Ala Ala Ala Ala Arg Ser Gly161 165 171 176Gly Tyr Gly Pro Gly Ser Gly Gln Gln Gly Pro Gly Gly Ala Gly Gln Gln Gly Pro Gly181 186 191 196Gly Leu Gly Pro Tyr Gly Pro Gly Ala Ala Ala Ala Ala Ala Gly Ser Tyr Gly Pro Arg201 206 211 216Ala Gly Gln Gln Arg Pro Gly Ala Gly Gln Gln Gly Pro Gly Ala Gly Gln Gln Gly Pro221 226 231 236Gly Gly Ala Gly Gln Arg Gly Pro Tyr Gly Pro Gly Ala Ala Ala Ala Ala Ala Ala Ala241 246 251 256Gly Gly Tyr Gly Gln Gln Gly Pro Gly Ala Gly Gln Gln Gly Pro Gly Val Gly Gln Gln261 266 271 276Gly Pro Gly Gly Ala Gly Gln Arg Gly Pro Tyr Gly Pro Gly Ala Ala Ala Ala Ala Ala281 286 291 296Ala Ala Gly Gly Tyr Gly Pro Arg Tyr Gly Gln Gln Gly Pro Gly Ala Gly Pro Tyr Gly301 306 311 316Pro Gly Ala Gly Ala Thr Ala Ala Ala Ala Gly Gly Tyr Gly Pro Gly Ala Gly Gln Gln321 326 331 336Gly Pro Arg Ser Gln Ala Pro Val Ala Ser Ala Ala Ala Ala Arg Leu Ser Ser Pro Gln341 346 351 356Ala Gly Ser Arg Val Ser Ser Ala Val Ser Thr Leu Val Ser Ser Gly Pro Thr Asn Pro361 366 371 376Ala Ser Leu Ser Asn Ala Ile Gly Ser Val Val Ser Gln Val Ser Ala Ser Asn Pro Gly381 386 391 396Leu Pro Ser Cys Asp Val Leu Val Gln Ala Leu Leu Glu Ile Val Ser Ala Leu Val His401 406 411 416Ile Leu Gly Ser Ser Ser Ile Gly Gln Ile Asn Tyr Ser Ala Ser Ser Gln Tyr Ala Arg421 426 431 436Leu Val Gly Gln Ser Ile Ala Gln Ala Leu Gly His His His His His His441 446 451 456
5.根据权利要求4所述悦目金蛛大壶状腺丝蛋白的人工基因-I重组蛋白的制备方法，其特征是以人工基因-I[MaSp2(2+1)]编码区段的核苷酸序列与质粒pET28制备成pET28-MaSp2(2+1)表达质粒，在无NaCL的LB中30-35℃培养到OD600为0.5，经IPTG诱导，破碎细胞后经Ni-NTA His-bind亲和层析柱和分子筛两次纯化后，得到纯化的重组蛋白。
6.一种编码悦目金蛛大壶状腺丝蛋白的人工基因-II重组蛋白氨基酸序列为Gly Gly Tyr Gly Pro Gly Ser Gly Gln Gln Gly Pro Gly Gly Ala Gly Gln Gln Gly Pro1 6 11 16Gly Gly Leu Gly Pro Tyr Gly Pro Gly Ala Ala Ala Ala Ala Ala Gly Ser Tyr Gly Pro21 26 31 36Arg Ala Gly Gln Gln Arg Pro Gly Ala Gly Gln Gln Gly Pro Gly Ala Gly Gln G1n Gly41 46 51 56Pro Gly Gly Ala Gly Gln Arg Gly Pro Tyr Gly Pro Gly Ala Ala Ala Ala Ala Ala Ala61 66 71 76Ala Gly Gly Tyr Gly Gln Gln Gly Pro Gly Ala Gly Gln Gln Gly Pro Gly Val Gly Gln81 86 91 96Gln Gly Pro Gly Gly Ala Gly Gln Arg Gly Pro Tyr Gly Pro Gly Ala Ala Ala Ala Ala101 106 111 116Ala Ala Ala Gly Gly Tyr Gly Pro Arg Tyr Gly Gln Gln Gly Pro Gly Ala Gly Pro Tyr121 126 131 136Gly Pro Gly Ala Gly Ala Thr Ala Ala Ala Ala Gly Gly Tyr Gly Pro Gly Ala Gly Gln141 146 151 156Gln Gly Pro Arg Ser Gln Ala Pro Val Ala Ser Ala Ala Ala Ala Ala Ala Arg Ser Gly161 165 171 176Gly Tyr Gly Pro Gly Ser Gly Gln Gln Gly Pro Gly Gly Ala Gly Gln Gln Gly Pro Gly181 186 191 196Gly Leu Gly Pro Tyr Gly Pro Gly Ala Ala Ala Ala Ala Ala Gly Ser Tyr Gly Pro Arg201 206 211 216Ala Gly Gln Gln Arg Pro Gly Ala Gly Gln Gln Gly Pro Gly Ala Gly Gln Gln Gly Pro221 226 231 236Gly Gly Ala Gly Gln Arg Gly Pro Tyr Gly Pro Gly Ala Ala Ala Ala Ala Ala Ala Ala241 246 251 256Gly Gly Tyr Gly Gln Gln Gly Pro Gly Ala Gly Gln Gln Gly Pro Gly Val Gly Gln Gln261 266 271 276Gly Pro Gly Gly Ala Gly Gln Arg Gly Pro Tyr Gly Pro Gly Ala Ala Ala Ala Ala Ala281 286 291 296Ala Ala Gly Gly Tyr Gly Pro Arg Tyr Gly Gln Gln Gly Pro Gly Ala Gly Pro Tyr Gly301 306 311 316Pro Gly Ala Gly Ala Thr Ala Ala Ala Ala Gly Gly Tyr Gly Pro Gly Ala Gly Gln Gln321 326 331 336Gly Pro Arg Ser Gln Ala Pro Val Ala Ser Ala Ala Ala Ala Ala Ala Arg Ser Gly Gly341 346 351 356Tyr Gly Pro Gly Ser Gly Gln Gln Gly Pro Gly Gly Ala Gly Gln Gln Gly Pro Gly Gly361 366 371 376Leu Gly Pro Tyr Gly Pro Gly Ala Ala Ala Ala Ala Ala Gly Ser Tyr Gly Pro Arg Ala381 386 391 396Gly Gln Gln Arg Pro Gly Ala Gly Gln Gln Gly Pro Gly Ala Gly Gln Gln Gly Pro Gly401 406 411 416Gly Ala Gly Gln Arg Gly Pro Tyr Gly Pro Gly Ala Ala Ala Ala Ala Ala Ala Ala Gly421 426 431 436Gly Tyr Gly Gln Gln Gly Pro Gly Ala Gly Gln Gln Gly Pro Gly Val Gly Gln Gln Gly441 446 451 456Pro Gly Gly Ala Gly Gln Arg Gly Pro Tyr Gly Pro Gly Ala Ala Ala Ala Ala Ala Ala461 466 471 476Ala Gly Gly Tyr Gly Pro Arg Tyr Gly Gln Gln Gly Pro Gly Ala Gly Pro Tyr Gly Pro481 486 491 496Gly Ala Gly Ala Thr Ala Ala Ala Ala Gly Gly Tyr Gly Pro Gly Ala Gly Gln Gln Gly501 506 511 516Pro Arg Ser Gln Ala Pro Val Ala Ser Ala Ala Ala Ala Arg Leu Ser Ser Pro Gln Ala521 526 531 536Gly Ser Arg Val Ser Ser Ala Val Ser Thr Leu Val Ser Ser Gly Pro Thr Asn Pro Ala541 546 551 556Ser Leu Ser Asn Ala Ile Gly Ser Val Val Ser Gln Val Ser Ala Ser Asn Pro Gly Leu561 566 57l 576Pro Ser Cys Asp Val Leu Val Gln Ala Leu Leu Glu Ile Val Ser Ala Leu Val His Ile581 585 591 596Leu Gly Ser Ser Ser Ile Gly Gln Ile Asn Tyr Ser Ala Ser Ser Gln Tyr Ala Arg Leu601 606 611 616Val Gly Gln Ser Ile Ala Gln Ala Leu Gly His His His His His His621 626 631 636
7.根据权利要求6所述悦目金蛛大壶状腺丝蛋白的人工基因-II重组蛋白蛋白制备方法，其特征是以人工基因-II[MaSp2(3+1)]编码区段的核苷酸序列与质粒pET28制备pET28-MaSp2(3+1)表达质粒，在无NaCL的LB中30-35℃培养到OD600为0.5，经IPTG诱导，破碎细胞后经Ni-NTA His-bind亲和层析柱和分子筛两次纯化后，得到纯化的重组蛋白。
8.根据权利要求1、4、6所述悦目金蛛大壶状腺丝蛋白在材料科学、医学、军事及航空航天等领域作为可降解的新型生物材料方面的应用。
全文摘要
本发明属于基因工程技术领域，所述的悦目金蛛(Argiope amoena)大壶状腺丝蛋白基因(Aa－MaSp2)的长度为918 bp，该序列由816bp的编码区和99 bp的3′端非翻译区组成。根据Aa－MaSp2基因序列推导的多肽由272个氨基酸组成。具有高强度、高弹性双重优良特性的蜘蛛拖丝是由大壶状腺丝蛋白构成的。本发明利用Aa－MaSp2基因克隆制备融合蛋白，并利用该蛋白免疫动物制备多克隆抗体。为使通过基因工程手段获得的人工大壶状腺丝蛋白分子量尽量接近与蜘蛛自然产大壶状腺丝蛋白分子，采用基因操作技术人工拼接了编码大壶状腺丝蛋白的人工基因－I和人工基因－II。通过基因工程生产的大壶状腺丝蛋白可开发为人工拖丝，强度和弹性俱佳的人工拖丝有望成为一种在材料科学、医学、军事及航空航天等领域有应用价值的可降解的新型生物材料。
文档编号C12N15/12GK1563085SQ200410014449
公开日2005年1月12日 申请日期2004年3月26日 优先权日2004年3月26日
发明者周开亚, 宋大祥, 潘鸿春 申请人:南京师范大学